Non-standard insulin analogues

ABSTRACT

An insulin analogue comprises a B-chain polypeptide containing a cyclohexanylalanine substitution at position B24 and optionally containing additional amino-acid substitutions at positions A8, B28, and/or B29. A proinsulin analogue or single-chain insulin analogue containing a B domain containing a cyclohexanylalanine substitution at position B24 and optionally containing additional amino-acid substitutions at positions A8, B28, and/or B29. The analogue may be an analogue of a mammalian insulin, such as human insulin. A nucleic acid encoding such an insulin analogue is also provided. A method of lowering the blood sugar of a patient comprises administering a physiologically effective amount of the insulin analogue or a physiologically acceptable salt thereof to a patient. A method of semi-synthesis using an unprotected octapeptide by means of modification of an endogenous tryptic site by non-standard amino-acid substitutions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of pending U.S. Provisional ApplicationNo. 61,507,324 filed on Jul. 13, 2011.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under cooperativeagreements awarded by the U.S. National Institutes of Health under grantnumbers DK40949 and DK074176. The U.S. government may have certainrights to the invention.

BACKGROUND OF THE INVENTION

This invention relates to polypeptide hormone analogues that exhibitsenhanced pharmaceutical properties, such as more rapid pharmacokineticsand/or augmented resistance to thermal fibrillation above roomtemperature. More particularly, this invention relates to insulinanalogues that are modified by the incorporation of non-standard aminoacids. Such non-standard sequences may optionally contain standardamino-acid substitutions at other sites in the A or B chains of aninsulin analogue.

The engineering of non-standard proteins, including therapeutic agentsand vaccines, may have broad medical and societal benefits. An exampleof a medical benefit would be optimization of the pharmacokineticproperties of a protein. An example of a further societal benefit wouldbe the engineering of proteins more refractory than standard proteinswith respect to degradation at or above room temperature for use inregions of the developing world where electricity and refrigeration arenot consistently available. An example of a therapeutic protein isprovided by insulin. Analogues of insulin containing non-standardamino-acid substitutions may in principle exhibit superior propertieswith respect to pharmacokinetics or resistance to thermal degradation.The challenge posed by the pharmacokinetics of insulin absorptionfollowing subcutaneous injection affects the ability of patients toachieve tight glycemic control and constrains the safety and performanceof insulin pumps. The challenge posed by its physical degradation isdeepened by the pending epidemic of diabetes mellitus in Africa andAsia. These issues are often coupled as modifications known in the artto accelerate absorption following subcutaneous injection usually worsenthe resistance of insulin to chemical and/or physical degradation.Because fibrillation poses the major route of degradation above roomtemperature, the design of fibrillation-resistant formulations mayenhance the safety and efficacy of insulin replacement therapy in suchchallenged regions. The present invention pertains to the use of aparticular class of non-standard amino acids—an aliphatic ring system asexemplified by Cyclohexanylalanine (Cha)—to modify and improve distinctproperties of insulin. During the past decade specific chemicalmodifications to the insulin molecule have been described thatselectively modify one or another particular property of the protein tofacilitate an application of interest. Whereas at the beginning of therecombinant DNA era (1980) wild-type human insulin was envisaged asbeing optimal for use in diverse therapeutic contexts, the broadclinical use of insulin analogues in the past decade suggests that asuite of non-standard analogs, each tailored to address a specific unmetneed, would provide significant medical and societal benefits.Substitution of one natural amino acid at a specific position in aprotein by another natural amino acid is well known in the art and isherein designated a standard substitution. Non-standard substitutions ininsulin offer the prospect of accelerated absorption without worseningof the resistance to degradation.

Administration of insulin has long been established as a treatment fordiabetes mellitus. Insulin is a small globular protein that plays acentral role in metabolism in vertebrates. Insulin contains two chains,an A chain, containing 21 residues, and a B chain containing 30residues. The hormone is stored in the pancreatic β-cell as aZn²⁺-stabilized hexamer, but functions as a Zn²⁺-free monomer in thebloodstream. Insulin is the product of a single-chain precursor,proinsulin, in which a connecting region (35 residues) links theC-terminal residue of B chain (residue B30) to the N-terminal residue ofthe A chain (FIG. 1A). Although the structure of proinsulin has not beendetermined, a variety of evidence indicates that it consists of aninsulin-like core and disordered connecting peptide (FIG. 1B). Formationof three specific disulfide bridges (A6-A11, A7-B7, and A20-B19; FIGS.1A and 1B) is thought to be coupled to oxidative folding of proinsulinin the rough endoplasmic reticulum (ER). Proinsulin assembles to formsoluble Zn²⁺-coordinated hexamers shortly after export from ER to theGolgi apparatus. Endoproteolytic digestion and conversion to insulinoccurs in immature secretory granules followed by morphologicalcondensation. Crystalline arrays of zinc insulin hexamers within maturestorage granules have been visualized by electron microscopy (EM). Thesequence of insulin is shown in schematic form in FIG. 1C. Individualresidues are indicated by the identity of the amino acid (typicallyusing a standard three-letter code), the chain and sequence position(typically as a superscript).

Aromatic side chains in insulin, as in globular proteins in general, mayengage in a variety of hydrophobic and weakly polar interactions,involving not only neighboring aromatic rings but also other sources ofpositive- or negative electrostatic potential. Examples includemain-chain carbonyl- and amide groups in peptide bonds. Hydrophobicpacking of aromatic side chains can occur within the core of proteinsand at non-polar interfaces between proteins. Such aromatic side chainscan be conserved among vertebrate proteins, reflecting their keycontributions to structure or function. An example of a natural aromaticamino acid is phenylalanine. Its aromatic ring system contains sixcarbons arranged as a planar hexagon. Aromaticity is a collectiveproperty of the binding arrangement among these six carbons, leading toπ electronic orbitals above and below the plane of the ring. These facesexhibit a partial negative electrostatic potential whereas the edge ofthe ring, containing five C—H moieties, exhibits a partial positiveelectrostatic potential. This asymmetric distribution of partial chargesgives rise to a quadrapole electrostatic moment and may participate inweakly polar interactions with other formal or partial charges in aprotein. An additional characteristic feature of an aromatic side chainsis its volume. Determinants of this volume include the topographiccontours of its five C—H moieties at the edges of the planar ring.Substitution of an aromatic ring system by a corresponding aliphaticring system would increase side-chain volume with loss of planarity andgain of one additional hydrogen atom at each carbon site (e.g.,substitution of each C—H element with trigonal hybridization by CH₂ withtetrahedral hybridization).

An example of a conserved aromatic residue in a therapeutic protein isprovided by phenylalanine at position B24 of the B chain of insulin(designated Phe^(B24)). This is one of three phenylalanine residues ininsulin (positions B1, B24, and B25). A structurally similar tyrosine isat position B26. The structural environment of Phe^(B24) in an insulinmonomer is shown in a ribbon model (FIG. 1D) and in a space-fillingmodel (FIG. 1E). Conserved among vertebrate insulins and insulin-likegrowth factors, the aromatic ring of Phe^(B24) packs against (but notwithin) the hydrophobic core to stabilize the super-secondary structureof the B-chain. Phe^(B24) lies at the classical receptor-binding surfaceand has been proposed to direct a change in conformation on receptorbinding.

The pharmacokinetic features of insulin absorption after subcutaneousinjection have been found to correlate with the rate of disassembly ofthe insulin hexamer. Although not wishing the present invention to beconstrained by theory, modifications to the insulin molecule that leadto accelerated disassembly of the insulin hexamer are thought to promotemore rapid absorption of insulin monomers and dimers from thesubcutaneous depot into the bloodstream. Phe^(B24) packs at the dimerinterface of insulin and so at three interfaces of an insulin hexamer.Its structural environment in the insulin monomer differs from itsstructural environment at these interfaces. In particular, thesurrounding volume available to the side chain of Phe^(B24) is larger inthe monomer than in the dimer or hexamer.

A major goal of insulin replacement therapy in patients with diabetesmellitus is tight control of the blood glucose concentration to preventits excursion above or below the normal range characteristic of healthyhuman subjects. Excursions below the normal range are associated withimmediate adrenergic or neuroglycopenic symptoms, which in severeepisodes lead to convulsions, coma, and death. Excursions above thenormal range are associated with increased long-term risk ofmicrovascular disease, including retinaphthy, blindness, and renalfailure. Because the pharmacokinetics of absorption of wild-type humaninsulin following subcutaneous injection is often too slow and tooprolonged relative to the physiological requirements of post-prandialmetabolic homeostasis, considerable efforts have been expended duringthe past 20 years to develop insulin analogues that exhibit more rapidabsorption with pharmacodynamic effects that are more rapid in onset andless prolonged in duration. Examples of such rapid-acting analoguesknown in the art are [Lys^(B28), pro^(B29)]-insulin (KP-insulin, theactive component of Humalog®), [Asp^(B28)]-insulin (Novalog®), and[Lys^(B3), Glu^(B29)]-insulin (Apidra®). Although widely used inclinical practice, these analogues exhibit two principal limitations.First, although their pharmacokinetic and pharmacodynamic profiles aremore rapid than those of wild-type insulin, they are not rapid enough inmany patients to optimize glycemic control or enable the safe andeffective use of algorithm-based insulin pumps (closed-loop systems).Second, the amino-acid substitutions in these analogues impair thethermodynamic stability of insulin and exacerbate its susceptibility tofibrillation above room temperature. Thus, the safety, efficacy, andreal-world convenience of these products have been limited by atrade-off between accelerated absorption and accelerated degradation.

Protein Engineering and the Mechanism of Insulin Absorption.

The major structural interface of the insulin hexamer is provided by ananti-parallel β-sheet at the dimerization surface. The componentβ-strands comprise residues B24-B28 and dimer-related residuesB24′-B28′; this segment has the amino-acid sequence FFYTP. The core ofthe β-sheet is provided by the three aromatic side chains Phe^(B24),Phe^(B25), and Tyr^(B26), which in the active insulin monomer alsocontact the insulin receptor. Substitutions known in the art to providerapid-acting and active insulin analogues occur at positions B28(Pro^(B28) in wild-type insulin) and flanking site B29 (Lys^(B29) inwild-type insulin). Standard amino-acid substitutions at core sites B24,B25, and B26 have not been employed in past design of insulin analoguesintended for the treatment of patients with diabetes mellitus since suchsubstitutions, as known in the art, typically impair biologicalactivity. Substitution of Phe^(B24) by Tyr, for example, impairsactivity by more than twentyfold despite its seemingly conservativecharacter. The importance of these invariant aromatic residues has beenhighlighted by the finding of genetic (germ-line) mutations at positionsB24 and B25 that cause diabetes mellitus in human patients.

Fibrillation, which is a serious concern in the manufacture, storage anduse of insulin and insulin analogues for the treatment of diabetesmellitus, is enhanced with higher temperature, lower pH, agitation, orthe presence of urea, guanidine, ethanol co-solvent, or hydrophobicsurfaces. Current US drug regulations demand that insulin be discardedif fibrillation occurs at a level of one percent or more. Becausefibrillation is enhanced at higher temperatures, patients with diabetesmellitus optimally must keep insulin refrigerated prior to use.Fibrillation of insulin or an insulin analogue can be a particularconcern for such patients utilizing an external insulin pump, in whichsmall amounts of insulin or insulin analogue are injected into thepatient's body at regular intervals. In such a usage, the insulin orinsulin analogue is not kept refrigerated within the pump apparatus, andfibrillation of insulin can result in blockage of the catheter used toinject insulin or insulin analogue into the body, potentially resultingin unpredictable fluctuations in blood glucose levels or even dangeroushyperglycemia. At least one recent report has indicated that insulinLispro (KP-insulin, an analogue in which residues B28 and B29 areinterchanged relative to their positions in wild-type human insulin;trade name Humalog®) may be particularly susceptible to fibrillation andresulting obstruction of insulin pump catheters. Insulin exhibits anincrease in degradation rate of 10-fold or more for each 10° C.increment in temperature above 25° C.; accordingly, guidelines call forstorage at temperatures <30° C. and preferably with refrigeration.

The present theory of protein fibrillation posits that the mechanism offibrillation proceeds via a partially folded intermediate state, whichin turn aggregates to form an amyloidogenic nucleus. In this theory, itis possible that amino-acid substitutions that stabilize the nativestate may or may not stabilize the partially folded intermediate stateand may or may not increase (or decrease) the free-energy barrierbetween the native state and the intermediate state. Therefore, thecurrent theory indicates that the tendency of a given amino-acidsubstitution in the insulin molecule to increase or decrease the risk offibrillation is highly unpredictable.

There is a need, therefore for an insulin analogue that displays morerapid hexamer disassembly while exhibiting at least a portion of theactivity of the corresponding wild-type insulin and maintaining at leasta portion of its chemical and/or physical stability.

SUMMARY OF THE INVENTION

It is, therefore, an aspect of the present invention to provide insulinanalogues that provide more rapid hexamer disassembly and henceaccelerated absorption following subcutaneous injection. The presentinvention addresses previous limitations for fast-acting insulinanalogues, namely, that they still do not act sufficiently quickly tooptimize glycemic control or enable use in implantable insulin pumps asthey are more susceptible to fibrillation than wild-type insulin. Theclaimed invention circumvents previous design restrictions, includingthose regarding substitution of Phe^(B24), through the incorporation ofa non-standard amino-acid substitution at position B24. The non-standardamino-acid side chain (Cyclohexanylalanine at position B24; Cha^(B24))markedly enhances rapidity of hexamer disassembly, the rate-limitingstep in insulin absorption in humans. This is achieved by substitutionof an aromatic amino-acid side chain by a non-aromatic analogue, whichis non-planar but of approximately similar size and shape toPhenylalanine, where the analogue then maintains at least a portion ofbiological activity of the corresponding insulin or insulin analoguecontaining the native aromatic side chain.

In general, the present invention provides an insulin analoguecomprising an insulin B-chain polypeptide containing at least onesubstitution selected from a cyclohexanylalanine substitution atposition B24 and a substitution at position B29 selected from the groupconsisting of norleucine, aminobutyric acid, aminopropionic acid,ornithine, diaminobutyric acid, and diaminopropionic acid. In anotherembodiment, the insulin analogue is a mammalian insulin analogue, suchas an analogue of human insulin. In one embodiment, the insulin analogueadditionally comprises substitution at position B28. In addition or inthe alternative, an insulin analogue may optionally comprise a Glusubstitution at position A8. In one particular set of embodiments, theB-chain polypeptide comprises an amino-acid sequence selected from thegroup consisting of SEQ. ID. NOS. 4-7, and 21 and polypeptides havingthree or fewer additional amino-acid substitutions thereof. In yetanother particular set of embodiments, designated single-chain insulinanalogs, the B-chain polypeptide is part of a single extendedpolypeptide of length 51-86 that comprises an amino-acid sequenceprovided in SEQ. ID. NO 8, and polypeptides having three or feweradditional amino-acid substitutions thereof.

In addition or in the alternative, the insulin analogue may contain anon-standard amino-acid substitution at position 29 of the B-chain. Inone particular example, the non-standard amino acid at B29 is norleucine(Nle). In another particular example, the non-standard amino acid at B29is ornithine (Orn).

Also provided is a nucleic acid encoding an insulin analogue comprisinga B-chain polypeptide that incorporates a non-standard amino acid atposition B24 or B29 or both. In one example, the non-standard amino acidis encoded by a stop codon, such as the nucleic acid sequence TAG. Anexpression vector may comprise such a nucleic acid and a host cell maycontain such an expression vector.

The invention also provides a method of lowering the blood sugar apatient. The method comprises administering a physiologically effectiveamount of an insulin analogue or a physiologically acceptable saltthereof to the patient, wherein the insulin analogue or aphysiologically acceptable salt thereof contains a B-chain polypeptideincorporating a Cyclohexanylalanine as described above. In oneembodiment, the Cyclohexanylalanine in the insulin analogue administeredto a patient is located at position B24. In still another embodiment,the insulin analogue is a mammalian insulin analogue, such as ananalogue of human insulin. In one particular set of embodiments, theB-chain polypeptide comprises an amino-acid sequence selected from thegroup consisting of SEQ. ID. NOS. 4-8, 21 and polypeptides having threeor fewer additional amino-acid substitutions thereof.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A is a schematic representation of the sequence of humanproinsulin including the A- and B-chains and the connecting region shownwith flanking dibasic cleavage sites (filled circles) and C-peptide(open circles).

FIG. 1B is a structural model of proinsulin, consisting of aninsulin-like moiety and a disordered connecting peptide (dashed line).

FIG. 1C is a schematic representation of the sequence of human insulinindicating the position of residue B24 in the B-chain.

FIG. 1D is a ribbon model of an insulin monomer showing aromatic residueof Phe^(B24) in relation to the three disulfide bridges. The adjoiningside chains of Leu^(B15) (arrow) and Phe^(B24) are shown. The A- andB-chain chains are otherwise shown in light and dark gray, respectively,and the sulfur atoms of cysteines as circles.

FIG. 1E is a space-filling model of insulin showing the Phe^(B24) sidechain within a pocket at the edge of the hydrophobic core.

FIG. 2A is a series of ball and stick (top) and space-filling (bottom)representations of Phenylalanine (Phe).

FIG. 2B is a series of ball and stick (top) and space-filling (bottom)representations of Cyclohexanylalanine (Cha).

FIG. 3 is a series of views of a structural model depicting the fit ofCha^(B24) within the structure of wild-type insulin. (FIGS. 3A and B)Wild-type insulin monomer (FIG. 3A) and dimer (FIG. 3B) as extractedfrom the crystal structure of the T₆ zinc insulin hexamer (ProteinDatabank accession code 4INS); (FIGS. 3C and D) Corresponding modelspredicting the fit of Cyclohexanylalanine in the variant monomer (FIG.3C) and dimer (FIG. 3D).

FIG. 4 is a graph showing the results of receptor-binding studies ofinsulin analogues. Relative activities for the B isoform of the insulinreceptor (IR-B) are determined by competitive binding assay in whichreceptor-bound ¹²⁵I-labeled human insulin is displaced by increasingconcentrations of human insulin (•) or its analogues: KP-insulin (▴) andCha^(B24)-KP-insulin (▾).

FIG. 5 provides 2D ¹H-NMR NOESY spectra of DKP-insulin (FIG. 5A),Cha^(B24)-DKP-insulin (FIG. 5B), and Cha^(B25)-DKP-insulin (FIG. 5C),each recorded at 700 MHz at 32° C. and pD 7.0. (Left) TOCSY spectrum ofaromatic region. Resonance assignments are as indicated. (Right) NOESYspectrum providing inter-proton contacts between aromatic protons(vertical axis) and aliphatic protons (horizontal axis). Empty box inFIG. 5B highlights absence of ring-current-shifted resonances due tonon-aromatic nature of Cha^(B24); arrows in FIG. 5A and FIG. 5C indicatecharacteristic upfield shift of Leu^(B15) methyl resonance due to ringcurrent of Phe^(B24). The disorder of Phe^(B25) on the surface of aninsulin monomer by contrast attenuates its ring-current effects.

FIG. 6A is a graph showing the results of receptor-binding studies ofwild type human insulin (▪), KP-insulin (•), Cha^(B24)-KP-insulin (▴) orGlu^(A8)-Cha^(B24)-KP-insulin (▾) using isolated insulin receptor(isoform B).

FIG. 6B is a graph showing the results of receptor-binding studies ofwild type human insulin (▪), KP-insulin (•), Cha^(B24)-KP-insulin (▴) orGlu^(A8)-Cha^(B24)-KP-insulin (▾) using human IGF-1 receptor.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed an insulin analogue that provides amore rapid rate of hexamer disassembly where the analogue then maintainsat least a portion of biological activity of the correspondingunmodified insulin or insulin analogue.

The present invention pertains to non-standard modifications at positionB24 to improve the properties of insulin with respect to rapidity ofabsorption following subcutaneous injection. In one instance thenon-standard amino acid lacks aromaticity and its associated asymmetricdistribution of partial positive and negative charges as demonstrated bysubstitution of the non-planar aliphatic ring system ofcyclohexanylalanine. Loss of planarity in a non-aromatic ring system isassociated with a change in its topographical contours and an increasein side-chain volume (FIG. 2B) relative to phenylalanine (FIG. 2A).

In one embodiment, the present invention provides an insulin analoguethat provides more rapid hexamer disassembly by substitution ofphenylalanine at position B24 by a non-standard amino acid. In oneparticular embodiment the more rapid hexamer disassembly is directed bysubstitution of cyclohexanylalanine at position B24. The presentinvention is not limited, however, to human insulin and its analogues.It is also envisioned that these substitutions may also be made inanimal insulins such as porcine, bovine, equine, and canine insulins, byway of non-limiting examples.

It has also been discovered that Cha^(B24)-KP-insulin, when formulatedin Lilly Diluent and following subcutaneous injection in a male Lewisrat rendered diabetic by streptozotocin, will direct a reduction inblood glucose concentration with a potency similar to that of KP-insulinin the same formulation.

In addition or in the alternative, the insulin analogue of the presentinvention may contain a non-standard amino-acid substitution at position29 of the B chain, which is lysine (Lys) in wild-type insulin. In oneparticular example, the non-standard amino acid at B29 is norleucine(Nle). In another particular example, the non-standard amino acid at B29is ornithine (Orn).

Furthermore, in view of the similarity between human and animalinsulins, and use in the past of animal insulins in human patients withdiabetes mellitus, it is also envisioned that other minor modificationsin the sequence of insulin may be introduced, especially thosesubstitutions considered “conservative.” For example, additionalsubstitutions of amino acids may be made within groups of amino acidswith similar side chains, without departing from the present invention.These include the neutral hydrophobic amino acids: Alanine (Ala or A),Valine (Val or V), Leucine (Leu or L), Isoleucine (Ile or I), Proline(Pro or P), Tryptophan (Trp or W), Phenylalanine (Phe or F) andMethionine (Met or M). Likewise, the neutral polar amino acids may besubstituted for each other within their group of Glycine (Gly or G),Serine (Ser or S), Threonine (Thr or T), Tyrosine (Tyr or Y), Cysteine(Cys or C), Glutamine (Glu or Q), and Asparagine (Asn or N). Basic aminoacids are considered to include Lysine (Lys or K), Arginine (Arg or R)and Histidine (His or H). Acidic amino acids are Aspartic acid (Asp orD) and Glutamic acid (Glu or E). Unless noted otherwise or whereverobvious from the context, the amino acids noted herein should beconsidered to be L-amino acids.

Standard amino acids may also be substituted by non-standard amino acidsbelong to the same chemical class. By way of non-limiting example, thebasic side chain Lys may be replaced by basic amino acids of shorterside-chain length (Ornithine, Diaminobutyric acid, or Diaminopropionicacid). Lys may also be replaced by the neutral aliphatic isostereNorleucine (Nle), which may in turn be substituted by analoguescontaining shorter aliphatic side chains (Aminobutyric acid orAminopropionic acid).

In one example, the insulin analogue of the present invention containsthree or fewer conservative substitutions other than the cyclicaliphatic substitution of the present invention.

As used in this specification and the claims, various amino acids ininsulin or an insulin analogue may be noted by the amino-acid residue inquestion, followed by the position of the amino acid, optionally insuperscript. The position of the amino acid in question includes the A-or B chain of insulin where the substitution is located. Thus, Phe^(B24)denotes a phenylalanine at the twenty-fourth amino acid of the B chainof insulin. Unless noted otherwise or wherever obvious from the context,the location of substitutions should be understood to be relative to andin the context of human insulin. Aromatic and non-aromatic rings differin planarity, reflecting the presence (Phe) or absence (Cha) of πelectrons as illustrated in front and side views of Phenylalanine (FIG.2A) relative to Cyclohexanylalanine (FIG. 2B).

Although not wishing to be constrained by theory, the present inventionenvisions that modifications at B24 that alter the weakly polarcharacter of the ring system and/or enlarge its topographical contourswould more readily be accommodated in the insulin monomer than at thedimer interface and so be associated with accelerated disassembly. Inparticular, because the dimer interface is characterized by multiplearomatic-aromatic interactions involving Phe^(B24) and six otheraromatic rings (Tyr^(B16), Phe^(B25), Tyr^(B26), and theirsymmetry-related partners), the present invention further envisions thatloss of aromaticity at position B24 would in general accelerate thedisassembly of insulin hexamers and further accelerate the disassemblyof variant hexamers containing destabilizing mutations elsewhere in thedimer- or trimer interface. Although the three-dimensional structure ofa Cha^(B24) variant of human insulin has not been determined, insightmay be gained from rigid-body modeling based on the crystal structure ofwild-type insulin (FIG. 3). A molecular model depicting the packing ofCha^(B24) within an insulin monomer is shown in FIG. 3C relative to thewild-type Phe^(B24) as shown in FIG. 3A. A molecular model depicting thepacking of Cha^(B24) within an insulin dimer interface is shown in FIG.3D relative to the wild-type Phe^(B24) as shown in FIG. 3A.

The phenylalanine at B24 is an invariant amino acid in functionalinsulin and contains an aromatic side chain. The biological importanceof Phe^(B24) in insulin is indicated by a clinical mutation (Ser^(B24))causing human diabetes mellitus. As illustrated in FIGS. 1D and 1E, andwhile not wishing to be bound by theory, Phe^(B24) is believed to packat the edge of a hydrophobic core at the classical receptor bindingsurface. The models are based on a crystallographic protomer (2-Znmolecule 1; Protein Databank identifier 4INS). Lying within theC-terminal β-strand of the B-chain (residues B24-B28), Phe^(B24) adjoinsthe central α-helix (residues B9-B19). In the insulin monomer one faceand edge of the aromatic ring sit within a shallow pocket defined byLeu^(B15) and Cys^(B19); the other face and edge are exposed to solvent(FIG. 1E). This pocket is in part surrounded by main-chain carbonyl andamide groups and so creates a complex and asymmetric electrostaticenvironment with irregular and loose steric borders. In the insulindimer, and within each of the three dimer interfaces of the insulinhexamer, the side chain of Phe^(B24) packs within a more tightlycontained spatial environment as part of a cluster of eight aromaticrings per dimer interface (TyrB16, Phe^(B24), Phe^(B25), Tyr^(B26) andtheir dimer-related mates). Irrespective of theory, substitution of thearomatic ring of Phe^(B24) by a cyclic aliphatic ring of the same numberof carbon atoms, but differing in its volume, stereo-electronicproperties, and lack of planarity, provides an opportunity to preservegeneral hydrophobic packing within the dimer interface of the insulinhexamer while imposing distinct spatial packing constraints andperturbing the asymmetric electrostatic environment of the wild-typearomatic ring.

The present invention pertains to a non-standard modification atposition B24 to improve the properties of insulin or insulin analogueswith respect to rapidity of absorption following subcutaneous injection.In one instance the non-standard amino acid lacks aromaticity and itsassociated asymmetric distribution of partial positive and negativecharges as demonstrated by substitution of the non-planar aliphatic ringsystem of Cyclohexanylalanine. Loss of planarity in a non-aromatic ringsystem is associated with a change in topographical contours and anincrease in side-chain volume (FIG. 2B) relative to phenylalanine (FIG.2A). In other instances the non-standard amino-acid substitution at B24is accompanied by a non-standard substitution at position B29 or bythree or fewer standard substitutions elsewhere in the A- or B chains.

It is envisioned that the substitutions of the present invention may bemade in any of a number of existing insulin analogues. For example, thecyclic aliphatic side chain (Cha) substitution at position B24 providedherein may be made in insulin analogues such as insulin Lispro([Lys^(B28), Pro^(B29)]-insulin, herein abbreviated KP-insulin), insulinAspart (Asp^(B28)-insulin), other modified insulins or insulinanalogues, or within various pharmaceutical formulations, such asregular insulin, NPH insulin, lente insulin or ultralente insulin, inaddition to human insulin. Insulin Aspart contains an Asp^(B28)substitution and is sold as Novalog® whereas insulin Lispro containsLys^(B28) and Pro^(B29) substitutions and is known as and sold under thename Humalog®. These analogues are described in U.S. Pat. Nos. 5,149,777and 5,474,978, the disclosures of which are hereby incorporated byreference herein. These analogues are each known as fast-actinginsulins.

The amino-acid sequence of human proinsulin is provided, for comparativepurposes, as SEQ ID NO: 1.

(human proinsulin) SEQ ID NO: 1Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr-Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr- Cys-Asn

The amino-acid sequence of the A chain of human insulin is provided asSEQ ID NO: 2.

(human A chain) SEQ ID NO: 2Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn

The amino-acid sequence of the B chain of human insulin is provided asSEQ ID NO: 3.

(human B chain) SEQ ID NO: 3Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Lys-Thr

The amino-acid sequence of a B chain of human insulin may be modifiedwith a substitution of a Cyclohexanylalanine (Cha) at position B24. Anexample of such a sequence is provided as SEQ. ID. NO 4.

SEQ ID NO: 4 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-Xaa₄-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Xaa₁-Phe-Tyr-Thr-Xaa₂-Xaa₃-Thr[Xaa₁ is Cha; Xaa₂ is Asp, Pro, Lys, or Arg; Xaa₃is Lys, Pro, or Ala; and Xaa₄ is His or Asp]

Substitution of a Cha at position B24 may optionally be combined withnon-standard substitutions at position B29 as provided in SEQ. ID. NO 5.

SEQ ID NO: 5 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-Xaa₃-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Xaa₁-Phe-Tyr-Thr-Pro-Xaa₂-Thr

[Xaa₁ is Cha; Xaa₂ is Asp, Pro; Xaa₂ is Ornithine, Diaminobutyric acid,Diaminoproprionic acid, Norleucine, Aminobutric acid, or Aminoproprionicacid; and Xaa₃ is His or Asp]

Further combinations of other substitutions are also within the scope ofthe present invention. It is also envisioned that the substitutionsand/or additions of the present invention may also be combined withsubstitutions of prior known insulin analogues. For example, theamino-acid sequence of an analogue of the B chain of human insulincontaining the Lys^(B28) and Pro^(B29) substitutions of insulin Lispro,in which the Cha^(B24) substitution may also be introduced, is providedas SEQ ID NO: 6.

SEQ ID NO: 6 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Xaa₁-Phe-Tyr-Thr-Lys-Pro-Thr [Xaa₁ is Cha]

Similarly, the amino-acid sequence of an analogue of the B chain ofhuman insulin containing the Asp^(B28) substitution of insulin Aspart,in which the Cha^(B24) substitution may also be introduced, is providedas SEQ ID NO: 7.

SEQ ID NO: 7 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Xaa₁-Phe-Tyr-Thr-Asp-Lys-Thr [Xaa₁ is Cha]

A Cha^(B24) substitution may also be introduced in combination withother insulin analogue substitutions such as analogues of human insulincontaining His substitutions at residues A4, A8 and/or B1 as describedmore fully in co-pending International Application No. PCT/US07/00320and U.S. application Ser. No. 12/160,187, the disclosures of which areincorporated by reference herein. For example, the Cha^(B24)substitution may be present with His^(A8) and/or His^(B1) substitutionsin a single-chain insulin analogue or proinsulin analogue having theamino-acid sequence represented by SEQ ID NO: 8,

SEQ ID NO: 8 Xaa₁-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Xaa₈-Phe-Xaa₂-Thr-Xaa₃-Xaa₄-Thr-Xaa₅-Gly-Ile-Val-Xaa₆-Gln-Cys-Cys-Xaa₇-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu- Glu-Asn-Tyr-Cys-Asn;

wherein Xaa₁ is His or Phe; wherein Xaa₂ is Tyr or Phe, Xaa₃ is Pro,Lys, or Asp; wherein Xaa₄ is Lys or Pro; wherein Xaa₆ is His or Glu;wherein Xaa₇ is His or Thr; wherein Xaa₅ is 0-35 of any amino acid or abreak in the amino-acid chain; and wherein Xaa₈ is Cha; and furtherwherein at least one substitution selected from the group of thefollowing amino-acid substitutions is present:

-   -   Xaa₁ is His; and    -   Xaa₇ is His; and    -   Xaa₆ and Xaa₇ together are His.

A Cyclohexanylalanine substitution at B24 and/or two amino acid additionmay also be introduced into a single-chain insulin analogue as disclosedin co-pending U.S. patent application Ser. No. 12/419,169, thedisclosure of which is incorporated by reference herein.

In still another embodiment, the B-chain insulin analogue polypeptidecontains a Lysine at position B3, Glutamic acid at position B29, andCyclohexanylalanine at position B24 as provided as SEQ ID NO: 9.

SEQ ID NO: 9 Phe-Val-Lys-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Xaa₁-Phe-Tyr-Thr-Pro-Glu-Thr.

Wherein Xaa₁ is Cyclohexanylalanine.

Cyclohexanylalanine was introduced within an engineered insulin monomerof native activity, designated KP-insulin, which contains thesubstitutions Lys^(B28) (K) and Pro^(B29) (P). These two substitutionson the surface of the B-chain are believed to impede formation of dimersand hexamers but be compatible with hexamer assembly in the presence ofzinc ions and a phenolic preservative. KP-insulin is the activeingredient of Humalog®, currently in clinical use as a rapid-actinginsulin analogue formulation. The sequence of the B-chain polypeptidefor this variant of KP-insulin is provided as SEQ ID NO: 6.Cyclohexanylalanine was also introduced at position B24 (SEQ ID NO: 21),and separately at position B25 (SEQ ID NO: 22) as a control analogue,within an engineered insulin monomer of enhanced activity, designatedDKP-insulin, which contains the substitution Asp^(B10) (D) in additionto the KP substitutions Lys^(B28) (K) and Pro^(B29) (P) in accordancewith the general scheme provided in SEQ. ID. NO 4. Cha^(B24) was alsointroduced into non-standard human insulin analogues containing eitherOrnithine or Norleucine at position B29 in accordance with the generalscheme provided in SEQ. ID. NO 5.

Analogues of KP-insulin and DKP-insulin were prepared bytrypsin-catalyzed semi-synthesis and purified by high-performance liquidchromatography (Mirmira, R. G., and Tager, H. S., 1989. J. Biol. Chem.264: 6349-6354). This protocol employs (i) a synthetic octapeptiderepresenting residues (N)-GF*FYTKPT (including modified residue (F*) and“KP” substitutions (underlined); SEQ ID NO: 12) and (ii) truncatedanalogue des-octapeptide[B23-B30]-insulin or, in the case of DKP-insulinanalogues, Asp^(B10)-des-octapeptide[B23-B30]-insulin (SEQ ID NO: 10).Because the octapeptide differs from the wild-type B23-B30 sequence(GF*FYTPKT; SEQ ID NO: 11) by interchange of Pro^(B28) and Lys^(B29)(italics), protection of the lysine ε-amino group is not required duringtrypsin treatment. In brief, des-octapeptide (15 mg) and octapeptide (15mg) were dissolved in a mixture of dimethylacetamide/1,4-butandiol/0.2 MTris acetate (pH 8) containing 10 mM calcium acetate and 1 mM ethylenediamine tetra-acetic acid (EDTA) (35:35:30, v/v, 0.4 mL). The final pHwas adjusted to 7.0 with 10 μL of N-methylmorpholine. The solution wascooled to 12° C., and 1.5 mg of TPCK-trypsin was added and incubated for2 days at 12° C. An additional 1.5 mg of trypsin was added after 24 hr.The reaction was acidified with 0.1% trifluoroacetic acid and purifiedby preparative reverse-phase HPLC (C4). Mass spectrometry usingmatrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF;Applied Biosystems, Foster City, Calif.) in each case gave expectedvalues (not shown). The general protocol for solid-phase synthesis is asdescribed (Merrifield et al., 1982. Biochemistry 21: 5020-5031).9-fluoren-9-yl-methoxy-carbonyl (F-moc)-protected phenylalanineanalogues were purchased from Chem-Impex International (Wood Dale,Ill.).

The above protocol was also employed to prepare analogues of humaninsulin containing Ornithine or Norleucine at position B29 and tointroduce Cha^(B24) in these respective contexts. The method ofpreparation of these analogues exploits non-standard amino-acidsubstitutions at position 29 to eliminate the tryptic site ordinarilypresent within the C-terminal octapeptide of the B chain (i.e., betweenLys^(B29) and Thr^(B30)) while maintaining a Proline at position 28.Pro^(B28) contributes to the stability of the dimer interface within theinsulin hexamer, and so this method of preparation providesnear-isosteric models of wild-type insulin in which other modificationsmay conveniently be incorporated without the need for cumbersomeside-chain protection.

Circular dichroism (CD) spectra were obtained at 4° C. and/or 25° C.using an Aviv spectropolarimeter (Weiss et al., Biochemistry 39:15429-15440). Samples contained ca. 25 μM DKP-insulin or analogues in 50mM potassium phosphate (pH 7.4); samples were diluted to 5 μM forguanidine-induced denaturation studies at 25° C. To extract freeenergies of unfolding, denaturation transitions were fitted bynon-linear least squares to a two-state model as described by Sosnick etal., Methods Enzymol. 317: 393-409. In brief, CD data θ(x), where xindicates the concentration of denaturant, were fitted by a nonlinearleast-squares program according to

${\theta (x)} = \frac{\theta_{A} + {\theta_{B}^{{({{{- \Delta}\; G_{H_{2}O}^{o}} - {mx}})}/{RT}}}}{1 + ^{{- {({{\Delta \; G_{H_{2}O}^{o}} - {mx}})}}/{RT}}}$

where x is the concentration of guanidine and where θ_(A) and θ_(B) arebaseline values in the native and unfolded states. Baselines wereapproximated by pre- and post-transition lines θ_(A)(x)=θ_(A) ^(H) ²^(O)+m_(A)x and θ_(B)(x)=θ_(B) ^(H) ² ^(O)+m_(B)x. The m values obtainedin fitting the variant unfolding transitions are lower than the m valueobtained in fitting the wild-type unfolding curve. To test whether thisdifference and apparent change in ΔG_(u) result from an inability tomeasure the CD signal from the fully unfolded state, simulations wereperformed in which the data were extrapolated to plateau CD values athigher concentrations of guanidine; essentially identical estimates ofΔG_(u) and m were obtained.

Relative activity is defined as the ratio of the hormone-receptordissociation constants of analogue to wild-type human insulin, asmeasured by a competitive displacement assay using ¹²⁵I-human insulin.Microtiter strip plates (Nunc Maxisorb) were incubated overnight at 4°C. with AU5 IgG (100 μl/well of 40 mg/ml in phosphate-buffered saline).Binding data were analyzed by a two-site sequential model. Data werecorrected for nonspecific binding (amount of radioactivity remainingmembrane associated in the presence of 1 μM human insulin. In all assaysthe percentage of tracer bound in the absence of competing ligand wasless than 15% to avoid ligand-depletion artifacts. Representative dataare provided in FIG. 4.

To assess hypoglycemic potencies of KP-insulin (or DKP-insulin)analogues relative to KP-insulin or wild-type insulin in vivo, maleLewis rats (mean body mass ˜300 grams) were rendered diabetic bytreatment with streptozotocin. (This model provides a probe of potencybut not degree of acceleration of pharmacokinetics as (i) wild-typeinsulin, KP-insulin, and Asp^(B28)-insulin exhibit similar patterns ofeffects of blood glucose concentration and (ii) these patterns areunaffected by the presence of absence of zinc ions in the formulation ata stoichiometry sufficient to ensure assembly of insulin hexamers).Protein solutions containing wild-type human insulin, insulin analogues,or buffer alone (protein-free sterile diluent obtained from Eli Lillyand Co.; composed of 16 mg glycerin, 1.6 mg meta-cresol, 0.65 mg phenol,and 3.8 mg sodium phosphate PH 7.4) were injected subcutaneously, andresulting changes in blood glucose were monitored by serial measurementsusing a clinical glucometer (Hypoguard Advance Micro-Draw meter). Toensure uniformity of formulation, insulin analogues were eachre-purified by reverse-phase high-performance liquid chromatography(rp-HPLC), dried to powder, dissolved in diluent at the same maximumprotein concentration (300 μg/mL) and re-quantitative by analytical C4rp-HPLC; dilutions were made using the above buffer. Rats were injectedsubcutaneously at time t=0 with 20 μg insulin in 100 μl of buffer per300 g rat. This dose corresponds to ca. 67 μg/kg body weight, whichcorresponds in international units (IU) to 2 IU/kg body weight.Dose-response studies of KP-insulin indicated that at this dose anear-maximal rate of glucose disposal during the first hour followinginjection was achieved. Five rats were studied in the group receivingCha^(B24)-KP-insulin (SEQ ID NOS: 2 and 6), and five different rats werestudied in the control group receiving KP-insulin (SEQ ID NOS: 2 and20); these rats were randomly selected from a colony of 30 diabeticrats. The two groups exhibited similar mean blood glucose concentrationsat the start of the experiment. Blood was obtained from clipped tip ofthe tail at time 0 and every 10 minutes up to 90 min. The efficacy ofinsulin action to reduce blood glucose concentration was calculatedusing the change in concentration over time (using least-mean squaresand initial region of linear fall) divided by the concentration ofinsulin injected. The initial rate of change in blood glucoseconcentration in the group receiving KP-insulin was −127.1±24.6 mg/dl/h(mean±standard error of the mean); the initial rate of change in thegroup receiving Cha^(B24)-KP-insulin was −113.5±21.7 mg/dl/h. Anydifferences were not statistically significant. These data thus suggestthat the biological potency of Cha^(B24)-KP-insulin is equivalent tothat of KP-insulin in a zinc hexamer formulation.

The kinetic stability of insulin analogue hexamers was assessed at 25°C. relative to that of the wild-type human insulin hexamer as a cobalt(Co²⁺) complex in the presence of 2.2 cobalt ions per hexamer and 50 mMphenol in a buffer consisting of 10 Tris-HCl (pH 7.4). The assay, amodification of the procedure of Beals et al. (Birnbaum, D. T.,Kilcomons, M. A., DeFelippis, M. R., & Beals, J. M. Assembly anddissociation of human insulin and Lys^(B28), Pro^(B29)-insulin hexamers:a comparison study. Pharm Res. 14, 25-36 (1997)), employs opticalabsorbance at 500-700 nm to monitor the R₆-hexamer-specific d-dtransitions characteristic of tetrahedral cobalt ion coordination.Although the solution at equilibrium contains a predominance of cobaltinsulin hexamers or cobalt insulin analogue hexamers, this equilibriumis characterized by opposing rates of insulin assembly and disassembly.To initiate the assay, the solution is made 2 mM inethylene-diamine-tetra-acetic acid (EDTA) to sequester free cobalt ions.The time course of decay of the R₆-specific absorption band on additionof EDTA provides an estimate of the rate of hexamer disassembly. Whereaswild-type insulin (SEQ ID NOS: 2 and 3) exhibited a time constant of419±51 seconds, KP-insulin (SEQ ID NOS: 2 and 20) exhibited a timeconstant of 114±13 seconds in accordance with its acceleratedpharmacokinatics. Strikingly, the time constant for Cha^(B24)-KP-insulin(SEQ ID NOS: 2 and 6) was found to be 49±5 seconds, predicting a furtheracceleration of pharmacokinetics in human patients. Stated differently,Cha^(B24)-KP-insulin is almost as accelerated in its disassemblyrelative to KP-insulin, as KP-insulin is accelerated relative to wildtype human insulin.

The far-ultraviolet circular dichroism (CD) spectrum of the Cha^(B24)analogue is similar to those of the parent analogues. Modified B24residues were introduced within the context of KP-insulin (SEQ ID NO:6), DKP-insulin (SEQ ID NO: 21), and non-standard analogues of humaninsulin in which Lys^(B29) was substituted by Ornithine or Norleucine(SEQ ID NO: 5). Activity values shown are based on ratio ofhormone-receptor dissociation constants relative to human insulin; theactivity of human insulin is thus 1.0 by definition. Standard errors inthe activity values were in general less than 25%. Free energies ofunfolding (ΔG_(u)) at 25° C. were estimated based on a two-state modelas extrapolated to zero denaturant concentration. Lag time indicatestime (in days) required for initiation of protein fibrillation on gentleagitation at 30° C. in zinc-free phosphate-buffered saline (pH 7.4).

The baseline thermodynamic stability of KP-insulin, as inferred from atwo-state model of denaturation at 25° C., is 3.0±0.1 kcal/mole.CD-detected guanidine denaturation studies indicate that the Cha^(B24)substitution is associated with a small decrement in thermodynamicstability in the context of KP-insulin (ΔΔG_(u) 0.3±0.2 kcal/mole) andin the context of DKP-insulin (ΔΔG_(u) 0.4±0.2 kcal/mole). Nonetheless,the physical stability of the Cha^(B24) KP analogue was found to besimilar to or greater than that of KP-insulin as evaluated in triplicateduring incubation in 300 μM phosphate-buffered saline (PBS) at pH 7.4 at30° C. under gentle agitation. The samples were observed for 20 days oruntil signs of precipitation or frosting of the glass vial wereobserved. Whereas the three tubes of KP-insulin became cloudy in 10, 13,and 16 days, respectively, the three tubes of Cha^(B24)-KP-insulinbecame cloudy in 13, 15, and 20 days. These data exhibit a trend towardgreater resistance to physical degradation by the Cha^(B24) analogue.

Dissociation constants (K_(d)) were determined as described by Whittakerand Whittaker (2005. J. Biol. Chem. 280: 20932-20936), by a competitivedisplacement assay using ¹²⁵I-Tyr^(A14)-insulin (kindly provided byNovo-Nordisk) and the purified and solubilized insulin receptor (isoformB or A) in a microtiter plate antibody capture assay with minormodification; transfected receptors were tagged at their C-terminus by atriple repeat of the FLAG epitope (DYKDDDDK; SEQ ID NO:23) andmicrotiter plates were coated by anti-FLAG M2 monoclonal antibody(Sigma). The percentage of tracer bound in the absence of competingligand was less than 15% to avoid ligand-depletion artifacts. Bindingdata were analyzed by non-linear regression using a heterologouscompetition model (Wang, 1995, FEBS Lett. 360: 111-114) to obtaindissociation constants. Results are provided in Table 1 (Cha^(B24)KP-insulin analogue relative to KP-insulin) and Table 2(Cha^(B25)-DKP-insulin relative to DKP-insulin); dissociation constantsare provided in units of nanomolar. (The two studies were conducted ondifferent dates with different preparations of insulin receptor (IRisoform B; IR-B) and IGF receptor (IGF-1R) and so are tabulatedindependently). The Cha^(B24) modification of KP-insulin reduces IR-Breceptor-binding affinities by between twofold and threefold; such smallreductions are typically associated with native or near-nativehypoglycemic potencies in vivo as demonstrated herein in diabetic Lewisrats. No significant increase was observed in the cross-binding ofCha^(B24)-KP-insulin to IGF-1R. The Cha^(B24) modification ofDKP-insulin reduces IR-B receptor-binding affinities by less thantwofold; a trend toward increased cross-binding to IGF-1R was observednear the limit of statistical significance. Cha^(B24)-DKP-insulin wasnot tested in rats. The affinity of Cha^(B25)-DKP-insulin for IR-B wasmarkedly impaired (binding to IR-B decreased by more than tenfold) inaccordance with classical structure-activity relationships in insulin.The distinct site-specific effects of a Phe→Cha substitution (welltolerated at B24 but not at B25) presumably reflect the differentstructural roles of these aromatic side chains at the hormone-receptorinterface.

TABLE 1 Binding of Insulin Analogues to Insulin Receptor and IGFReceptor Protein IR-B binding IGF-1R binding insulin 0.045 ± 0.007 nM5.1 ± 0.8 nM KP-insulin 0.093 ± 0.012 nM 5.0 ± 0.6 nMCha^(B24)-KP-insulin 0.171 ± 0.022 nM 4.3 ± 0.7 nM IR-B, B isoform ofthe insulin receptor; IGF-1R, Type 1 IGF receptor

TABLE 2 Binding of Insulin Analogues to Insulin Receptor and IGFReceptor Protein IR-B binding IGF-1R binding DKP-insulin 0.020 ± 0.003nM 3.1 ± 0.51 nM Cha^(B24)-DKP-insulin 0.032 ± 0.005 nM 1.4 ± 0.22 nMCha^(B25)-DKP-insulin 0.350 ± 0.050 nM ND IR-B, B isoform of the insulinreceptor. ND, not determined.

The binding affinities of analogues containing the non-standard aminoacids Ornithine or Norleucine at position B29 were similarly tested,both with and without a Cha substitution at B24. Results are provided inTable 3 as a percentage of the binding affinity of human insulin forhuman insulin receptor isoform A (hIR-A), human insulin receptor isoformB (hIR-B), and human IGF receptor (hIGF-1R); asterisks indicate valuesindistinguishable from 100% (wild-type) given experimental error.Whereas Orn^(B29) has similar binding affinities for each receptor aswild-type insulin (asterisks), Nle^(B29) confers a small decrease inaffinity for hIR-B and IGF-1R relative to wild type insulin. An analoguecontaining Orn^(B29) in combination with Cha^(B24), however, haddecreased binding affinity for both isoforms of insulin receptor andslightly increased affinity for hIGF-1R (possibly non-significant givenexperimental error). The Cha^(B24), Nle^(B29) analogue had similarbinding affinity for hIGF-1R as the Nle^(B29) only analogue, but haddecreased binding affinity for hIR-B. We highlight the modesty of thesechanges in affinity as the observed range of in vitro hIR affinities arein each case in accordance with expected in vivo hypoglycemic potenciessimilar to those of wild-type insulin (i.e., as tested in a rat model);similarly, the range of in vitro IGF-1R affinities are within the rangeof relative affinities exhibited by insulin analogs in current clinicaluse. These data provide evidence that substitutions Orn^(B29) andNle^(B29) have utility in semi-synthetic insulin formulations intendedfor therapeutic use, either alone or in combination with second-sitemodifications such as Cha^(B24).

TABLE 3 Relative Binding Affinity of Insulin Analogues to InsulinReceptor and IGF Receptor Protein hIR-A binding hIR-B binding hIGF-1Rbinding Insulin 100  100  100  Cha^(B24), Nle^(B29) 50 36 62 Cha^(B24),Orn^(B29) 58 53 134* Nle^(B29) ND 67 61 Orn^(B29)  95* 105* 115* hIR-A,A isoform of human insulin receptor; hIR-B, B isoform of human insulinreceptor; hIGF-1R, human IGF receptor; ND, not determined; percenterrors are in general less than 20% of the values given. Asterisksindicate values whose 95% confidence intervals include 100 and so may beindistinguishable from wild-type.

Two-dimensional ¹H-NMR spectra have been obtained of Cha^(B24) andCha^(B25) analogues of DKP-insulin (FIG. 5). Whereas the spectrum ofCha^(B25)-DKP-insulin is similar to that of DKP-insulin in accordancewith past studies suggesting that the ensemble-averaged aromaticring-current effects of Phe^(B25) are negligible in an insulin monomer,aliphatic substitution of Phe^(B24) leads to attenuation ofPhe^(B24)-related ring current effects. Qualitative interpretation ofthese spectra is nonetheless suggestive of native-like structures.Further evidence for native-like packing of Cha^(B24) was provided bythe crystallization of [Cha^(B24), Orn^(B29)]-insulin under conditionsthat routinely yields crystals of wild-type zinc insulin hexamers.

Insulin analogues additionally containing a Cha^(B24) substitution in aLys^(B28), Pro^(B29) analogue (SEQ ID NO: 6) were created with either awild type A-chain (SEQ ID NO: 2) or an A-chain containing a Glu^(A8)substitution (SEQ ID NO: 19). The results of competitive displacementassays using ¹²⁵I-labeled insulin as a tracer assays for human insulinreceptor isoform B and human type 1 insulin-like growth factor receptor(IGFR-1) are provided in FIGS. 6A and 6B, respectively. As shown in FIG.6A, the affinities of Cha^(B24)-KP-insulin (▴) andGlu^(A8)-Cha^(B24)-KP-insulin (▾) are similar to that of KP-insulin (•).Similarly, cross-binding of Cha^(B24)-KP-insulin andGlu^(A8)-Cha^(B24)-KP-insulin to IGFR-1 is within the margin of errorfor that of KP insulin (FIG. 6B).

CD spectra of Cha^(B24)-KP-insulin and Glu^(A8)-Cha^(B24)-KP-insulinresemble that of KP-insulin. 2D ¹H-NMR spectra of Cha^(B24)-KP-insulinretain native-like long-range NOEs but differ in pattern of chemicalshifts in accord with the loss of the Phe^(B24) ring current. Wemeasured the free energies of unfolding of Cha^(B24)-KP-insulin andGlu^(A8)-Cha^(B24)-KP-insulin relative to KP-insulin in a zinc-freebuffer at pH 7.4 and 25° C. (10 mM potassium phosphate and 50 mM KCl).This assay utilized CD detection of guanidine-induced denaturation asprobed at 222 nm. Values of ΔG_(u) were estimated on the basis of a2-state model. For Cha^(B24)-KP-insulin a possible slight decrease instability was seen that was within experimental error (ΔΔG_(u)0.1±0.2kcal/mole); for Glu^(A8)-Cha^(B24)-KP-insulin an increase was observed(ΔΔG_(u)0.5±0.2 kcal/mole). This assay predicts resistance to chemicaldegradation similar to or greater than that of Humalog®.

The respective fibrillation lag times of KP-insulin,Cha^(B24)-KP-insulin and Glu^(A8)-Cha^(B24)-KP-insulin under monomericconditions at 45° C. were investigated. The proteins were made 60 μM inphosphate-buffered saline at pH 7.4 in the absence of zinc ions.Fibrillation was detected by enhancement of Thioflavin T (ThT)fluorescence and onset of cloudiness in the solution. Whereas KP-insulin(N=3 vials) formed fibrils within 2 days, Cha^(B24)-KP-insulin (N=3vials) formed fibrils on day 4; solutions ofGlu^(A8)-Cha^(B24)-KP-insulin (N=2 vials) were formed fibrils on day 7.These data strongly suggest that the analogues provided by the claimedinvention will exhibit physical stabilities at least as great asHumalog® or greater.

The EDTA sequestration assay described above was also used exploitsthese spectroscopic features as follows. At time t=0 a molar excess ofEDTA is added to a solution of R₆ insulin hexamers or insulin analoghexamers. Although EDTA does not itself attack the hexamer to strip itof metal ions, any Co²⁺ ions released in the course of transient hexamerdisassembly become trapped by the chelator and thus unavailable forreassembly. The rate of disappearance of the blue color (the tetrahedrald-d optical transition at 574 nm of the R-specific insulin-bound Co²⁺)thus provides an optical signature of the kinetics of hexamerdisassembly.

Respective exponential dissociation curves yield half-lives of 419±51sec (wild-type insulin), 113±13 sec (KP-insulin), and 49±5 sec(Cha^(B24)-KP-insulin). These differences are dramatic. Similar findingswere observed in recent studies of Glu^(A8)-Cha^(B24)-KP-insulin;indeed, its half life was 50% shorter than that of Cha^(B24)-KP-insulin,indicating that the stabilizing A-chain substitution Glu^(A8) (on thehexamer surface distant from the dimer interface) does not compromise,and may further accelerate, its rate of disassembly relative toCha^(B24)-KP-insulin. Because diffusion of zinc ions from thesubcutaneous depot is analogous to in vitro sequestration of cobalt ionsin the assay, these findings predict that Cha^(B24)-KP-insulin andGlu^(A8)-Cha^(B24)-KP-insulin will exhibit ultra-rapid PK/PD properties.

Cha^(B24)-KP-insulin was tested in 2 pigs and exhibited similar potency(consistent with the rat studies) and a trend toward ultra-rapid PD.Late t_(1/2) _(max) values of 211±11 (Humalog®) and 172±13 min(Cha^(B24)-KP-insulin) were observed (p=0.20). Further, a 2-foldreduction was seen in the tail of insulin action (AUC above baselineinfusion rate 4 mg/kg/min) between 3-5 hours post-injection which almostachieved statistical significance (p=0.07) despite the limited samplesize.

An individual pig whose response to Humalog® was discovered to beunusually slow (initial time to half-maximal PD (initial t_(1/2) _(max)) 81 min) was used to test the PD of the Cha^(B24) analogues. Although asingle individual, this pig was of potential interest as a model for thevariability in PK/PD often observed among human patients in whomanalogous half-maximal PD times as prolonged as 90 min have beendocumented. Remarkably, in this pig, Cha^(B24)-KP-insulin andGlu^(A8)-Cha^(B24)-KP-insulin exhibited initial t_(1/2) _(max) times of62 and 49 min, respectively; the more rapid PD ofGlu^(A8)-Cha^(B24)-KP-insulin is in accordance with the EDTAsequestration assay.

A method for treating a patient comprises administering an insulinanalogue containing a Cha-substituted Phe or additional amino-acidsubstitutions in the A or B chain as known in the art or describedherein. In one example, the Cha-substituted insulin analogue is aninsulin analogue containing Cha at position B24 in the context ofKP-insulin. In another example, Cha^(B24) is substituted within humaninsulin analogues containing non-standard modifications at position B29(Ornithine or Norleucine). It is yet another aspect of the presentinvention that use of non-standard amino-acid substitutions enables arapid and efficient method of preparation of insulin analogues bytrypsin-mediated semi-synthesis using unprotected octapeptides.

In still another example, the insulin analogue is administered by anexternal or implantable insulin pump. An insulin analogue of the presentinvention may also contain other modifications, such as a tether betweenthe C-terminus of the B-chain and the N-terminus of the A-chain asdescribed more fully in co-pending U.S. patent application Ser. No.12/419,169, the disclosure of which is incorporated by reference herein.

A pharamaceutical composition may comprise such insulin analogues andwhich may optionally include zinc. Zinc ions may be included in such acomposition at a level of a molar ratio of between 2.2 and 3.0 perhexamer of the insulin analogue. In such a formulation, theconcentration of the insulin analogue would typically be between about0.1 and about 3 mM; concentrations up to 3 mM may be used in thereservoir of an insulin pump. Modifications of meal-time insulinanalogues may be formulated as described for (a) “regular” formulationsof Humulin® (Eli Lilly and Co.), Humalog® (Eli Lilly and Co.), Novalin®(Novo-Nordisk), and Novalog® (Novo-Nordisk) and other rapid-actinginsulin formulations currently approved for human use, (b) “NPH”formulations of the above and other insulin analogues, and (c) mixturesof such formulations.

Excipients may include glycerol, glycine, arginine, Tris, other buffersand salts, and anti-microbial preservatives such as phenol andmeta-cresol; the latter preservatives are known to enhance the stabilityof the insulin hexamer. Such a pharmaceutical composition may be used totreat a patient having diabetes mellitus or other medical condition byadministering a physiologically effective amount of the composition tothe patient.

A nucleic acid comprising a sequence that encodes a polypeptide encodingan insulin analogue containing a sequence encoding at least a B-chain ofinsulin with a Cyclohexanylalanine at position B24 is also envisioned.This can be accomplished through the introduction of a stop codon (suchas the amber codon, TAG) at position B24 in conjunction with asuppressor tRNA (an amber suppressor when an amber codon is used) and acorresponding tRNA synthetase, which incorporates a non-standard aminoacid into a polypeptide in response to the stop codon, as previouslydescribed (Furter, 1998, Protein Sci. 7:419-426; Xie et al., 2005,Methods. 36: 227-238). The particular sequence may depend on thepreferred codon usage of a species in which the nucleic-acid sequencewill be introduced. The nucleic acid may also encode other modificationsof wild-type insulin. The nucleic-acid sequence may encode a modified A-or B-chain sequence containing an unrelated substitution or extensionelsewhere in the polypeptide or modified proinsulin analogues. Forexample, an A-chain containing a Glu^(A8) substitution may be utilized.The nucleic acid may also be a portion of an expression vector, and thatvector may be inserted into a host cell such as a prokaryotic host celllike an E. coli cell line, or a eukaryotic cell line such as S.cereviciae or Pischia pastoris strain or cell line.

For example, it is envisioned that synthetic genes may be synthesized todirect the expression of a B-chain polypeptide in yeast Piscia pastorisand other microorganisms. The nucleotide sequence of a B-chainpolypeptide utilizing a stop codon at position B24 for the purpose ofincorporating a Cyclohexanylalanine at that position may be either ofthe following or variants thereof:

(a) with Human Codon Preferences: (SEQ ID NO: 15)TTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTGTGCGGGGAACGAGGCTAGTTCTACACACCCAAGACC(b) with Pichia Codon Preferences: (SEQ ID NO: 16)TTTGTTAACCAACATTTGTGTGGTTCTCATTTGGTTGAAGCTTTGTACTTGGTTTGTGGTGAAAGAGGTTAGTTTTACACTCCAAAGACT

Similarly, a full length pro-insulin cDNA having human codon preferencesand utilizing a stop codon at position B24 for the purpose ofincorporating Cyclohexanylalanine at that position may have the sequenceof SEQ. ID NO. 17.

(SEQ ID NO: 17) TTTGTGAACC AACACCTGTG CGGCTCACAC CTGGTGGAAGCTCTCTACCT AGTGTGCGGG GAACGAGGCT AGTTCTACACACCCAAGACC CGCCGGGAGG CAGAGGACCT GCAGGTGGGGCAGGTGGAGC TGGGCGGCGG CCCTGGTGCA GGCAGCCTGCAGCCCTTGGC CCTGGAGGGG TCCCTGCAGA AGCGTGGCATTGTGGAACAA TGCTGTACCA GCATCTGCTC CCTCTACCAG CTGGAGAACT ACTGCAACTA G

Likewise, a full-length human pro-insulin cDNA utilizing a stop codon atposition B24 for the purpose of incorporating a Cyclohexanylalanine atthat position and having codons preferred by P. pastoris may have thesequence of SEQ ID NO: 18.

(SEQ ID NO: 18) TTTGTTAACC AACATTTGTG TGGTTCTCAT TTGGTTGAAGCTTTGTACTT GGTTTGTGGT GAAAGAGGTT AGTTTTACACTCCAAAGACT AGAAGAGAAG CTGAAGATTT GCAAGTTGGTCAAGTTGAAT TGGGTGGTGG TCCAGGTGCT GGTTCTTTGCAACCATTGGC TTTGGAAGGT TCTTTGCAAA AGAGAGGTATTGTTGAACAA TGTTGTACTT CTATTTGTTC TTTGTACCAA TTGGAAAACT ACTGTAACTA A

Other variants of these sequences, encoding the same polypeptidesequence, are possible, given the synonyms in the genetic code.

Based upon the foregoing disclosure, it should now be apparent thatinsulin analogues provided will carry out the objects set forthhereinabove. Namely, these insulin analogues exhibit enhanced rates ofdisassembly of insulin hexamers while maintaining at least a fraction ofthe biological activity of wild-type insulin. It is, therefore, to beunderstood that any variations evident fall within the scope of theclaimed invention and thus, the selection of specific component elementscan be determined without departing from the spirit of the inventionherein disclosed and described.

The following literature is cited to demonstrate that the testing andassay methods described herein would be understood by one of ordinaryskill in the art.

-   Furter, R., 1998. Expansion of the genetic code: Site-directed    p-fluoro-phenylalanine incorporation in Escherichia coli. Protein    Sci. 7:419-426.-   Merrifield, R. B., Vizioli, L. D., and Boman, H. G. 1982. Synthesis    of the antibacterial peptide cecropin A (1-33). Biochemistry 21:    5020-5031.-   Mirmira, R. G., and Tager, H. S. 1989. Role of the phenylalanine B24    side chain in directing insulin interaction with its receptor:    Importance of main chain conformation. J. Biol. Chem. 264:    6349-6354.-   Sosnick, T. R., Fang, X., and Shelton, V. M. 2000. Application of    circular dichroism to study RNA folding transitions. Methods    Enzymol. 317: 393-409.-   Wang, Z. X. 1995. An exact mathematical expression for describing    competitive biding of two different ligands to a protein molecule    FEBS Lett. 360: 111-114.-   Weiss, M. A., Hua, Q. X., Jia, W., Chu, Y. C., Wang, R. Y., and    Katsoyannis, P. G. 2000. Hierarchiacal protein “un-design”:    insulin's intrachain disulfide bridge tethers a recognition α-helix.    Biochemistry 39: 15429-15440.-   Whittaker, J., and Whittaker, L. 2005. Characterization of the    functional insulin binding epitopes of the full length insulin    receptor. J. Biol. Chem. 280: 20932-20936.-   Xie, J. and Schultz, P. G. 2005. An expanding genetic code. Methods.    36: 227-238.

What is claimed is:
 1. An insulin analogue comprising an insulin B-chainpolypeptide containing at least one substitution selected from acyclohexanylalanine substitution at position B24 and a substitution atposition B29 selected from the group consisting of norleucine,aminobutyric acid, aminopropionic acid, ornithine, diaminobutyric acid,and diaminopropionic acid.
 2. The insulin analogue of claim 1, whereinthe at least one substitution is the cyclohexanylalanine substitution atposition B24.
 3. The insulin analogue of claim 1, wherein the at leastone substitution is a norleucine or ornithine substitution at positionB29.
 4. The insulin analogue of claim 1, wherein the insulin B-chainpolypeptide additionally comprises a substitution at position B28. 5.The insulin analogue of claim 4, wherein the insulin B-chain polypeptidecomprises substitutions at both positions B28 and B29.
 6. The insulinanalogue of any one of the preceding claims, wherein the insulin B-chainpolypeptide additionally comprises an insulin A-chain polypeptidecontaining a Glu substitution at position A8.
 7. The insulin analogue ofclaim 6, wherein the insulin B-chain polypeptide additionally comprisesa paired substitution selected from a Lysine substitution at positionB28 with a Proline substitution at position B29, or an Aspartic acidsubstitution at position B28 with a Proline substitution at positionB29.
 8. The insulin analogue of claim 1, wherein the B-chain polypeptidecomprises an amino-acid sequence selected from the group consisting ofSEQ. ID. NOS. 4-7 and
 21. 9. A polypeptide having an amino acid sequenceof SEQ ID NO:
 8. 10. A nucleic acid encoding an insulin analogueaccording to claim
 1. 11. An expression vector comprising the nucleicacid of claim
 10. 12. A host cell transformed with the expression vectorof claim
 11. 13. A method of lowering the blood sugar of a patientcomprising administering a physiologically effective amount of aninsulin analogue or a physiologically acceptable salt thereof to thepatient, wherein the insulin analogue or a physiologically acceptablesalt thereof contains a B-chain polypeptide incorporating at least onesubstitution selected from a cyclohexanylalanine substitution atposition B24 or a substitution at position B29 selected from the groupornithine, diaminobutyric acid, diaminopropionic acid, norleucine,aminobutyric acid, or aminopropionic acid.
 14. The method of claim 13,wherein the at least one substitution is a cyclohexanylalaninesubstitution at position B24.
 15. The method of claim 14, wherein theinsulin analogue or a physiologically acceptable salt thereofadditionally comprises one or more of a glutamic acid substitution atposition A8, a lysine substitution at position B28 and a prolinesubstitution at position B29.